Chromosomal rearrangements are difficult to observe in living cells. Several techniques, including some CRISPR-based methods, for making these observations have been reported, but these experimental designs have inherent challenges. Wang et al. now report a novel approach named CRISPR live-cell fluorescent in situ hybridization or “CRISPR LiveFISH.”
Wang et al. made fluorescent ribonucleoproteins (fRNPs) by combining 2 fluorescent components. The first was a fusion protein consisting of catalytically dead Cas9 fused to enhanced green fluorescent protein (dCas9-EGFP). The other was Cy® 3-labeled guide RNA. This guide RNA was itself made up of non-fluorescent tracrRNA combined with Cy 3-labeled crRNA. The guide RNA was designed to target a region of chromosome 3 that has approximately 500 repeats. Repetitive target sequences were shown to be the key to success. The researchers found that a large excess of target site DNA sequences caused stabilization of RNP complexes, so that the guide RNA was not degraded by intracellular RNase enzymes.
After electroporation of the fRNPs into cells and incubation, brightly fluorescent regions were visible at the target sites. The team observed that the Cy 3 fluorescence signal was over 4-fold stronger than the EGFP fluorescence, so they decided to use fluorescently labeled crRNA, rather than fluorescent Cas9, in their further experiments. They used numerous fluorophores on their crRNA molecules in the following experiments. Alt-R S.p. dCas9 Protein was used in some of these experiments.
The research team showed that they could target a repetitive region of chromosome 13 with dCas9 in complex with ATTO™ 565-labeled guide RNA. This region was successfully detected in amniotic fluid cells from normal and Patau syndrome patients. Next, by using 2 guide RNAs labeled with fluorophores of different colors, the scientists were able to visualize both repetitive regions (in chromosomes 3 and 13) simultaneously in various cell types, including human primary T lymphocytes.
The researchers next used a cell line stably expressing a fluorescent, double-strand break (DSB) sensor protein (a truncated form of 53BP1 fused to fluorescent Apple protein). They targeted a repetitive region with fRNP containing A488-labeled guide RNA. This repetitive region is 36 kb from the PPP1R2 gene, which they then targeted and cut via a non-fluorescent RNP. With this setup, they were able to observe the dynamics of fluorescent 53BP1-Apple recruitment and activity near the A488 label.
Since this experiment worked so well on a single chromosome, the researchers next studied chromosomal translocation by using fRNPs to target and label the repetitive regions in chromosomes 3 and 13, while also using non-fluorescent RNPs to target and cut nearby genes. The 2 different colors on the 2 cut chromosomes moved close together and stayed together for several hours, which appeared to represent chromosomal translocation. This was verified by sequencing.
Next, the researchers decided to try to image RNA and DNA simultaneously. Since repetitive sequences were the key to their success in previous experiments, they chose to use cell line, U2OS 2-6-3, which contains repetitive sequences of both RNA (known as MS2) and DNA (the LacO sequence). The dCas9 used in previous experiments cannot target RNA, so another Cas protein, Cas13d, was used. This enzyme is a member of the Cas13 family of enzymes, which cut RNA instead of DNA. When Cas13 enzymes are catalytically inactivated, they do not cut RNA but can still be targeted to RNA. The researchers used catalytically deactivated Cas13d in an fRNP complex to target and thus detect MS2 RNA while at the same time using a dCas9 fRNP of another fluorescent color to target and thus detect LacO DNA. The researchers were able to detect the repetitive RNA and DNA sequences simultaneously, allowing them to observe RNA transcription dynamics.
Wang et al. concluded that the CRISPR LiveFISH technique is a great way to study the dynamics of chromosomal and RNA events. This technique depends on the proximity of a repetitive region of either DNA or RNA, but the research team pointed out that more than 60% of protein-coding genes are within 100 kb of a repetitive stretch of DNA. Therefore, CRISPR LiveFISH may have many applications both alone and in combination with other CRISPR-mediated approaches to studying nuclear events.
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